O6-Methylguanine-DNA methyltransferase (MGMT) is a highly promising target for improving the efficacy of alkylating agents in the treatment of brain tumors and other cancers. Recently, our laboratory provided the first data that human MGMT protein is phosphorylated at tyrosine and serine/threonines and that phosphorylation inhibits its activity. Two cellular protein kinases that phosphorylate these amino acids in the MGMT protein were also characterized. The overall objective of this proposal is to define the role of protein phosphorylation in MGMT function and to assess its significance to improve glioma therapy. We hypothesize that multisite phosphorylation of MGMT controls its catalytic activity, and protein stability in human gliomas. We postulate that the net phosphorylation state of tumor MGMT profoundly affects its function and, in turn the sensitivity of gliomas to alkylating agents. The Specific Aims to be pursued in this 4-year project are (1) to identify the in vivo phosphorylation sites of human MGMT and define the contribution of each phosphorylation to MGMT's DNA repair activity, (2) to characterize the phosphorylation-dependent turn-over of the MGMT protein through the ubiquitin-proteolytic pathway, and (3) to generate phospho-specific antibodies against the MGMT protein, assess the levels of phospho-MGMT in glioma specimens, cell lines, and to correlate it with the catalytic activity of MGMT and drug sensitivity. The structural impact of phosphorylations on the DNA binding domain of MGMT, and the consequent alterations in MGMT activity towards O6-methylguanine, and O6-benzylguanine will also be examined as a part of the first Specific Aim. Changes in MGMT phosphorylation during the cell cycle progression will be determined by using the phospho-specific antibodies. These original studies promise to yield novel and critical information on the physiological regulation of MGMT, and are likely to provide new directions for improved glioma therapy and MGMT-targeted cancer therapies.